aal conjugated to biotin  (Vector Laboratories)

Journal: Cells

Article Title: Fucosylated Proteome Profiling Identifies a Fucosylated, Non-Ribosomal, Stress-Responsive Species of Ribosomal Protein S3

doi: 10.3390/cells10061310

Figure Lengend Snippet: UEAI recognizes intracellular proteins. ( A ) WM793 cells were fixed and permeabilized with Triton X-100 or left unpermeabilized. Cells were subsequently stained with DAPI nuclear stain, FITC-AAL, FITC-UEAI, or no lectin as a control. Cell were visualized by IF for qualitative lectin staining (left panel). For quantification, WM793 and A375 cells were stained as above and analyzed by flow cytometry (right panel; MFI: Mean Fluorescence Intensity (of UEA1 per cell)). * p = 0.006 ( B ) Cells transduced and selected with lentivirus containing Cas9/no guide (ng) or Cas9/sgSLC35C1 (gC1) were grown in normal growth media and harvested for protein lysate. Lysates were probed with AAL and UEAI lectin to determine global levels of Golgi-dependent fucosylation. ( C ) WM793 cells were cultured for three days in 2-fluorofucose (2-FF, FUTi, 250 µM), L-fucose (L-fuc, 250 µM), or control. Cells were harvested for protein lysate and blotted for global AAL and UEAI levels.

Article Snippet: UEAI, AAL, UEAI-488, AAL-488, UEAI-biotin, AAL-biotin were purchased from Vector Laboratories (Burlingame, CA, USA).

Techniques: Staining, Flow Cytometry, Fluorescence, Cell Culture

Journal: Cells

Article Title: Fucosylated Proteome Profiling Identifies a Fucosylated, Non-Ribosomal, Stress-Responsive Species of Ribosomal Protein S3

doi: 10.3390/cells10061310

Figure Lengend Snippet: RPS3 is fucosylated in vitro and in vivo. ( A ) RPS3 is recognized by UEAI lectin by agarose-bound lectin pull-down (PD). Cell lines IPC298, MelJuso, and SK-MEL-119 were cultured in growth media and harvested for protein lysate. Lysates were incubated with bead (agarose bead control), AAL (AAL-agarose), or UEAI (UEAI-agarose) to bind fucosylated proteins. UEAI-recognized RPS3 in all three cell lines. ( B ) Liver, muscle, brain, and skin samples were harvested from a C57BL/6 mouse and lysed using a Dounce homogenizer. Lysates were subjected to UEAI PD to isolate UEAI-recognized proteins. RPS3 was recognized by UEAI in all tissue types. LE: long exposure. ( C ) Cells transduced and selected with lentivirus containing Cas9/no guide (ng) or Cas9/sgSLC35C1 (gC1) were grown in normal growth media and harvested for protein lysate ( B). Lysate were subjected to UEAI PD to isolate UEAI-recognized proteins. Knockout of the Golgi fucose transporter SLC35C1 did not affect the ability of UEAI to recognize RPS3. ( D ) Cells expressing myc-DDK (FLAG)-tagged RPS3 (RPS3-mDDK) were harvested for FLAG IP and Western blot. The membrane was probed with mouse anti-FLAG/anti-mouse-680 and UEAI-biotin/strep-800 to assess direct recognition of RPS3 by UEAI (signal overlap by LI-COR imaging). UEAI recognizes RPS3. ( E ) Cells were grown in normal growth media and harvested for UEAI PD. The lectin PD was supplemented with either no sugar (control), L-fucose (Fuc), glucose (Glc), rhamnose (Rham), or galactose (Gal) at 1 M final concentration. Analysis by Western blot showed that fucose and rhamnose interfere with UEAI lectin recognition of RPS3, whereas glucose and galactose did not affect lectin binding. ( F ) Cells grown in the presence of labeled fucose were analyzed to assess labeling of RPS3. Labeled lysates or unlabeled lysates (control) were subjected to neutravidin pull-down (Neu-PD) to isolate proteins that integrated the labeled fucose. RPS3 did integrate the labeled fucose. β-actin, a non-fucosylated protein, did not show any integration of the labeled fucose (negative control).

Article Snippet: UEAI, AAL, UEAI-488, AAL-488, UEAI-biotin, AAL-biotin were purchased from Vector Laboratories (Burlingame, CA, USA).

Techniques: In Vitro, In Vivo, Cell Culture, Incubation, Knock-Out, Expressing, Western Blot, Imaging, Concentration Assay, Binding Assay, Labeling, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: GOLPH3 Regulates EGFR in T98G Glioblastoma Cells by Modulating Its Glycosylation and Ubiquitylation

doi: 10.3390/ijms21228880

Figure Lengend Snippet: The knockdown of GOLPH3 perturbs the glycosylation processing of EGFR in T98G cells. ( A ) Detergent-soluble extracts were prepared from the indicated cells grown in 6-well plates, and samples were either left untreated (lanes 1 and 2), treated with Neuraminidase (lanes 3 and 4), or treated with PNGase F (lanes 5 and 6), followed by SDS-PAGE and immunoblot analysis using antibodies to detect the proteins indicated on the right. The immunoblot signal of anti-β-actin was used as the loading control. The position of molecular mass markers is indicated on the left. ( B ) Detergent-soluble extracts were prepared from the indicated cells, followed by immunoprecipitation (IP) of EGFR. Immunoprecipitated EGFR was analyzed by SDS-PAGE followed by either immunoblot (IB) to EGFR or lectin blot (LB) using the lectins indicated in the right: Sambucus nigra lectin (SNA-I), Aleuria aurantia lectin (AAL), and Peanut agglutinin (PNA). The position of a molecular mass marker is indicated on the left. ( C ) Densitometry quantification of the immunoblot or lectin blot signal as shown in B. Bars represent the mean ± standard deviation ( n = 3; * p < 0.05; ** p < 0.01).

Article Snippet: The biotin-conjugated Aleuria aurantia lectin (AAL; cat # B-1395) was from Vector Labs (Burlingame, CA, USA), and biotin-conjugated Sambucus nigra lectin (SNA-I; cat # BA-6802-1) and Arachis hypogaea lectin (PNA; cat # BA-2301-1) were from EY Laboratories (San Mateo, CA, USA).

Techniques: SDS Page, Western Blot, Immunoprecipitation, Marker, Standard Deviation